The pathogenicity of Vibrio cholerae, which infects mucosal surfaces in the small intestine, is believed to be a coordinated and multifactorial process. Unfortunately, knowledge of which genes are expressed during infection, as well as the regulatory proteins and host signals that govern their regulation, is rudimentary. A novel gene reporter system has been constructed for identifying in vivo induced genes of V. cholerae, and has been used to identify the vie (V. cholerae in vivo expressed) operon. vie is predicted to encode three two-component signal transduction proteins, including a sensor and two response regulators, the first such system described in V. cholerae. vie is unique among such systems both in its operon structure and its exclusive expression during infection. Based on analogy to other systems, it is hypothesized that the Vie proteins function by sensing environmental conditions in the host intestine and responding by regulating other genes. Characterization of vie thus provides a unique opportunity both to probe the host signals and bacterial regulatory proteins that control V. cholerae growth and pathogenicity as well as to allow investigation of vie-regulated genes. The goals of this research proposal are to characterize the vie operon and to identify and characterize vie-regulated genes. Mutations will be constructed in both vie and vie-regulated genes, and the effects of each on pathogenicity will be measured. The subcellular locations of the Vie proteins will be determined using immunochemical methods. The spatial and temporal expression patterns of the vie operon and vie-regulated genes will be determined during infection. This work may contribute to development of mucosal vaccines and to identification of new targets for therapies against mucosal pathogens.